An estimated 180 million people are infected with hepatitis C virus (HCV) globally and 3-4 million are newly infected each year and is the leading cause of death from liver disease in the United States. For those aware of their positive HCV status, treatment involves a year of clinic visits with an often poorly tolerated treatment regime and even so only about 50% of treated patients are cured. New drugs targeting the viral protein, NS3/4A, which is a bifunctional protease/helicase are in clinical trials. However, drug resistance is arising quickly, likely rendering many of these promising therapeutics obsolete before their time. We are proposing a new drug design strategy to avoid drug resistance, which we have successfully applied to HIV-1 protease and developed single-digit pM inhibitors that retain affinity against a panel of drug resistant viruses. We hypothesize that this strategy works and is applicable to the protease domain of HCV NS3/4A, since at a molecular level drug resistance is a change in the balance of molecular recognition events that selectively weakens inhibitor binding but maintains substrate recognition and cleavage. To understand and reduce the likelihood of drug resistance, the atomic details of substrate recognition need to be elucidated. Thus in this proposal we are characterizing the four diverse natural substrate complexes of NS3/4A, through a combination of crystal structures, molecular dynamics simulations and enzyme kinetics. The resulting structures will likely define a substrate envelope, which defines the region necessary for substrate recognition and will be compared with the binding of inhibitors and the known patterns of drug resistance. The HCV NS3/4A substrate envelope can then be used as an added constraint in the design, synthesis and assessment of novel NS3/4A protease inhibitors that will likely be less susceptible to HCV drug resistance.)